The present invention relates to substituted pyrimidine derivatives and their use in therapy. More specifically, but not exclusively, the invention relates to compounds that are capable of inhibiting one or more protein kinases, particularly polo-like kinases.
The Polo-like kinase family consists of key cell cycle regulatory enzymes with integral roles in controlling entry into and progression through mitosis. Many tumour cells express high levels of PLK1 and are responsive to antisense oligonucleotides targeting this protein.
Initiation of mitosis requires activation of M-phase promoting factor (MPF), i.e. the complex between CDK1 and B-type cyclins [Nurse, P. (1990) Nature, 344, 503-508]. The latter accumulate during the S and G2 phases of the cell cycle and promote the inhibitory phosphorylation of the MPF complex by WEE1, MIK1, and MYT1 kinases. At the end of the G2 phase, corresponding dephosphorylation by the dual-specificity phosphatase CDC25C triggers the activation of MPF [Nigg, E. A. (2001) Nat. Rev. Mol. Cell Biol., 2, 21-32]. In interphase, cyclin B localizes to the cytoplasm and becomes phosphorylated during prophase, followed by nuclear translocation. The nuclear accumulation of active MPF during prophase is thought to be important for initiating M-phase events [Takizawa, C. G. and Morgan, D. O. (2000) Curr. Opin. Cell Biol., 12, 658-665]. However, nuclear MPF is kept inactive by WEE1 unless counteracted by CDC25C. The phosphatase CDC25C itself, localized to the cytoplasm during interphase, accumulates in the nucleus in prophase. The nuclear entry of both cyclin B and CDC25C are promoted through phosphorylation by PLK1 [Roshak, A. K., Capper, E. A., Imburgia, C., Fornwald, J., Scott, G. and Marshall, L. A. (2000) Cell. Signalling, 12, 405-411]. This kinase is thus an important regulator of M-phase initiation.
In humans, there exist three closely related polo-like kinases (PLKs) [Glover, D. M., Hagan, I. M. and Tavares, A. A. (1998) Genes Dev., 12, 3777-3787]. They contain a highly homologous N-terminal catalytic kinase domain and their C-termini contain two or three conserved regions, the polo boxes. The function of the polo boxes remains incompletely understood but polo box-dependent PLK1 activity is required for proper metaphase/anaphase transition and cytokinesis [Seong, Y.-S., Kamijo, K., Lee, J.-S., Fernandez, E., Kuriyama, R., Miki, T. and Lee, K. S. (2002) J. Biol. Chem., 277, 32282-32293]. Of the three PLKs, PLK1 is the best characterized; it regulates a number of cell division cycle effects, including the onset of mitosis, DNA-damage checkpoint activation, regulation of the anaphase promoting complex, phosphorylation of the proteasome, and centrosome duplication and maturation. Mammalian PLK2 (also known as SNK) and PLK3 (also known as PRK and FNK) were originally shown to be immediate early gene products. PLK3 kinase activity appears to peak during late S and G2 phase. It is also activated during DNA damage checkpoint activation and severe oxidative stress. PLK3 also plays an important role in the regulation of microtubule dynamics and centrosome function in the cell and deregulated PLK3 expression results in cell cycle arrest and apoptosis [Wang, Q., Xie, S., Chen, J., Fukusawa, K., Naik, U., Traganos, F., Darzynkiewicz, Z., Jhanwar-Uniyal, M. and Dai, W. (2002) Mol. Cell. Biol., 22, 3450-3459]. PLK2 is the least-well understood homologue of the three PLKs. Both PLK2 and PLK3 may have additional important post-mitotic functions [Kauselmann, G., Weiler, M., Wulff, P., Jessberger, S., Konietzko, U., Scafidi, J., Staubli, U., Bereiter-Hahn, J., Strebhardt, K. and Kuhl, D. (1999) EMBO J., 18, 5528-5539].
The fact that human PLKs regulate some fundamental aspects of mitosis was shown by anti-PLK1 antibody microinjection of human tumour cells [Lane, H. A. and Nigg, E. A. (1996) J. Cell. Biol., 135, 1701-1713]. This treatment had no effect on DNA replication but impaired cell division. Cells were arrested in mitosis and showed abnormal distribution of condensed chromatin and monoastral microtubules nucleated from duplicated but unseparated centrosomes. By contrast, non-immortalized human cells arrested as single, mononucleated cells in G2. Moreover, when PLK1 function was blocked through adenovirus-mediated delivery of a dominant-negative gene, tumour-selective apoptosis in many tumour cell lines was observed, whereas again normal epithelial cells, although arrested in mitosis, escaped the mitotic catastrophe seen in tumour cells [Cogswell, J. P., Brown, C. E., Bisi, J. E. and Neill, S. D. (2000) Cell Growth Differ., 11, 615-623]. PLK1 activity is thus necessary for the functional maturation of centrosomes in late G2/early prophase and subsequent establishment of a bipolar spindle. Furthermore, these results suggest the presence in normal cells of a centrosome-maturation checkpoint that is sensitive to PLK1 impairment. Depletion of cellular PLK1 through the small interfering RNA (siRNA) technique also confirmed that this protein is required for multiple mitotic processes and completion of cytokinesis [Liu, X. and Erikson, R. L. (2003) Proc. Natl. Acad. Sci. USA, 100, 5789-5794]. A potential therapeutic rationale for PLK inhibition is also suggested by work with PLK1-specific antisense oligonucleotides, which were shown to induce growth inhibition in cancer cells both in vitro and in vivo [Spankuch-Schmitt, B., Wolf, G., Solbach, C., Loibl, S., Knecht, R., Stegmuller, M., von Minckwitz, G., Kaufmann, M. and Strebhardt, K. (2002) Oncogene, 21, 3162-3171]. Constitutive expression of PLK1 in mammalian cells was shown to lead to malignant transformation [Smith, M. R., Wilson, M. L., Hamanaka, R., Chase, D., Kung, H., Longo, D. L. and Ferris, D. K. (1997) Biochem. Biophys. Res. Commun., 234, 397-405]. Furthermore, overexpression of PLK1 is frequently observed in human tumours and PLK1 expression is of prognostic value for patients suffering from various types of tumours [Takahashi, T., Sano, B., Nagata, T., Kato, H., Sugiyama, Y., Kunieda, K., Kimura, M., Okano, Y. and Saji, S. (2003) Cancer Science, 94, 148-152; Tokumitsu, Y., Mori, M., Tanaka, S., Akazawa, K., Nakano, S. and Niho, Y. (1999) Int. J. Oncol., 15, 687-692; Wolf, G., Elez, R., Doermer, A., Holtrich, U., Ackermann, H., Stutte, H. J., Altmannsberger, H.-M., Rübsamen-Waigmann, H. and Strebhardt, K. (1997) Oncogene, 14, 543-549].
Although the therapeutic potential of pharmacological PLK inhibition has been appreciated [Kraker, A. J. and Booher, R. N. (1999) In Annual Reports in Medicinal Chemistry (Vol. 34) (Doherty, A. M., ed.), pp. 247-256, Academic Press], very little has been reported to date concerning small molecule PLK inhibitors that may be useful as drugs. One of the few biochemical PLK1 inhibitors characterized to date is scytonemin, a symmetric indolic marine natural product [Stevenson, C. S., Capper, E. A., Roshak, A. K., Marquez, B., Eichman, C., Jackson, J. R., Mattern, M., Gerwick, W. H., Jacobs, R. S. and Marshall, L. A. (2002) J. Pharmacol. Exp. Ther., 303, 858-866; Stevenson, C. S., Capper, E. A., Roshak, A. K., Marquez, B., Grace, K., Gerwick, W. H., Jacobs, R. S. and Marshall, L. A. (2002) Inflammation Research, 51, 112-114].
Scytonemin inhibits phosphorylation of CDC25C by recombinant PLK1 with an IC50 value of about 2 μM (at an ATP concentration of 10 μM). Inhibition is apparently reversible and the mechanism with respect to ATP of mixed-competitive mode. Similar potency against other protein serine/threonine- and dual specificity cell-cycle kinases, including MYT1, CHK1, CDK1/cyclin B, and PKC, was observed. Scytonemin showed pronounced anti-proliferative effects on various human cell lines in vitro. Further small molecule PLK inhibitors and their use in the treatment of proliferative disorders are described in International patent application WO2004/067000 in the name of Cyclacel Limited.
The present invention seeks to elucidate new small molecule PLK inhibitors. More specifically, the invention seeks to provide small molecule PLK inhibitors that have therapeutic applications in the treatment of a range of proliferative disorders.